Abstract
Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.
Highlights
Bovine ribonuclease A oligomerizes in the forms of dimers [1], trimers, tetramers, and higher order oligomers [2] during lyophilization from 40% acetic acid solutions
It was shown that the efficiency of double-stranded RNA (dsRNA) degradation by the RNase A dimers increases under the following conditions: (i) as the distance between the active sites of the dimer decreases; and (ii) as the orientation of the two RNA-binding patches of the oligomeric enzyme is more twisted around the molecules [7]
BS-RNase is endowed with several biological actions; its aspermatogenic, embryotoxic, immunosuppressive [15,16,17,18], and, in particular, antitumor (13, 18 –21) activities have been extensively studied over the years
Summary
Whereas the cytosolic ribonuclease inhibitor (cRI) [23,24,25] can block monomeric RNase A after its entrance into the cell, BS-RNase, because of its dimeric structure, would escape interaction with the inhibitor and be able to exert its enzymatic activity in the cell [26, 27] Based on these facts and taking into account that a significant activity against transformed cells was shown to be displayed in vitro and in vivo by RNase A dimerized by protein engineering [28], covalently cross-linked dimers and trimers of RNase A (29 –31), and a dimeric mutant of human pancreatic RNase [32], the question arose as to whether the various RNase A oligomers obtained by the lyophilization procedure and purified as described [2] might be endowed with similar biological actions. We have performed a series of in vitro and in vivo experiments, which demonstrate that RNase A dimers, trimers, and tetramers display aspermatogenic and antitumor activities that increase remarkably as a function of the oligomer mass and, at the same time, show a complete lack of embryotoxicity
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